Common toxicity problems with forages (Proceedings)

Nitrate Toxicity and Dirigibles

Sheep jump fences. There's nothing new about that. We usually blame the fence — for being old and in the way — or the sheep, for being ... well... a sheep. But did you know that a sheep's rumen contains hydrogen — the same hydrogen that fills dirigibles — and that this hydrogen is related to nitrate toxicity? You might say that this puts a whole new, uh, lift to this month's topic.

Hydrogen? This will take a little chemistry, so please bear with me.

A main feature of a healthy rumen is that it contains very little free oxygen. The technical term for a no-oxygen environment is ?anaerobic." Rumen microbes (bacteria, protozoa, and other assorted microscopic bugs and plant cells) function quite vigorously in this anaerobic environment. Most of these microbes, in fact, absolutely require a lifestyle without oxygen. They use metabolic processes that don't need much oxygen, and they produce all sorts of interesting products, like various types of acids, carbon dioxide, and lots of hydrogen ions, H+ . The normal level of hydrogen in a rumen may be fairly low by dirigible standards, but these rumen H+ ions are chemically very active. They will replace oxygen atoms in any vulnerable molecule and reduce its negative electrical charge.

Now things get interesting. If we make hay from some heavily fertilized orchardgrass at the end of a particularly cool and cloudy month, we may be suddenly faced with a real nutritional problem — nitrate toxicity — due in part to this hydrogen in the rumen.

Some background about nitrates: Nitrates are very soluble in water. Under normal conditions, plants absorb nitrogen into their roots as nitrates and then transport these nitrates up through the stem to the leaves. Meanwhile, leaf cells spend their days doing photosynthesis, which uses sunlight and carbon dioxide to create sugars (carbohydrates). When nitrates arrive in a leaf cell from the basement, leaf enzymes link them with carbohydrates to form real proteins, which the plant uses for all sorts of important functions. It's a clever system, and it usually works quite smoothly.

But photosynthesis requires sunlight. What happens if the days are heavily overcast for a period of weeks? (For example, in Oregon's Willamette Valley in March). Simple — without sunlight, leaf photosynthesis slows down, which, in turn restricts the manufacture of protein. But if the roots are still bathed in rich nitrate solutions in the soil, they dutifully keep pumping those nitrates up into the plant anyway. Because the leaf cells can't use those nitrates quickly, nitrate levels begin to rise in the leaves and stems.

Ironically, the standard laboratory analysis for crude protein doesn't detect this problem because this assay only looks for nitrogen, not true protein. Since nitrates and protein all contain some nitrogen, the crude protein assay cannot differ between them. Only a specialized test for nitrate can monitor nitrate levels in plants. Normal levels are less than 0.44% (dry matter basis). We start to worry when nitrate levels rise above this.

High nitrate levels don't harm plants, and anyway, the surplus is only temporary. As soon as the sun reappears, the leaves will make a new batch of carbohydrates and use up the backlog of nitrates, and all is well.

But if we make hay at the tail end of one of those cloudy periods, we may harvest plants at the wrong end of that cycle, plants with high levels of nitrates. If we then feed this hay to our livestock, well...

Even though the problem is called ?nitrate toxicity," the truth is that nitrates themselves are really not very toxic. So what's the problem?

The hydrogen in the rumen.

Let's back up and look at that the anaerobic environment in the rumen. Nitrates contain three oxygen atoms (NO3). Nitrates don't have a promising future in the rumen because in that highly reduced, low oxygen environment, the pesky hydrogen ions will quickly try to replace the oxygen atoms. The chemical sequence is that nitrate (NO3) is reduced to nitrite (NO2), which is then further reduced to ammonia (NH3). Rumen bacteria then use that ammonia in a similar way that plant cells use nitrate — they combine the ammonia with carbohydrates to produce true bacterial protein. Again, all is well.

But if for any reason the rumen doesn't contain enough soluble carbohydrates — for example if animals are fed mediocre hay containing lots of fiber and no grain or molasses — the situation becomes analogous to those green leaves: the bacterial cells become metabolically stymied. Without enough soluble carbohydrates for all that ammonia, the bacteria cannot manufacture bacterial protein. The entire chain of reactions then backs up, just like a traffic jam. As the rumen ammonia builds up, so does the product of the preceding step: the nitrites.

Unlike nitrates, nitrites are toxic. As the nitrite levels begin to rise in the rumen, some nitrite will cross the rumen wall into the blood. Nitrite then wreaks havoc in red blood cells. It changes hemoglobin from a molecule which readily combines with oxygen, to an abnormal molecule called ?methemoglobin," which is brown and doesn't combine with oxygen at all. Blood cells poisoned by nitrite don't carry oxygen. If enough blood cells are affected, the animal begins to suffer from oxygen deprivation. In other words, the animal's blood turns brown and it begins to suffocate.

But all is not lost. One method of coping with potential nitrate toxicity is to dump enough soluble carbohydrates into the rumen to feed the bacteria, which can then use those carbohydrates with the ammonia. Therefore, when faced with nitrate toxicity, feed corn, barley, oats, or any grain or molasses — nearly anything that contains lots of these carbohydrates. This will take the pressure off the metabolic traffic jam. The bacteria will begin making their proteins again, and nitrite levels in the rumen will quickly fall

Rumen hydrogen is a basic feature of ruminant life. But recently I read of an experimental procedure that could cause hydrogen levels to rise.

High levels of hydrogen in the rumen?!! Would this be the notorious "dirigible effect?" I have a vision of cloud-like sheep floating over fences...

Estrogen and the Single Plant

Fifty years ago in Australia, soon after subterranean clover (Trifolium subterraneum) became a popular forage there, farmers and scientists observed a dramatic decline of fertility in ewes grazing subclover pastures. The longer the sheep grazed these fields, the lower their fertility. They also observed occasional udder development and aberrant lactation in nonpregnant ewes and even wethers. The Australians called this problem "clover disease" and concluded that it was caused by a type of estrogen. But who would put estrogens in the rations of grazing sheep? It turns out that it was the plants themselves — they contained hormone-active chemicals called phytoestrogens.

Phyto = derived from or associated with plants. Estrogen = a steroid hormone with powerful physiological effects, especially on the reproduction organs. Some plants, particularly legumes, can contain phytoestrogen, sometimes at high levels, and there can be profound effects when these plants are consumed by livestock.

Before everyone runs out and pulls their sheep and cattle off perfectly good legume pastures, I must add that most legumes are safe most of the time. Only some species of legumes contain enough phytoestrogens to cause problems. Also, some perfectly good legumes, which normally are quite safe for livestock, will only produce phytoestrogens when they suffer from certain kinds of stress. But read on.

Phytoestrogens in legumes come in two chemical flavors: the isoflavones and the coumestans. These two families of estrogen-like compounds are derived from different biochemical pathways. The isoflavones are usually found in the true clovers — subclover, red clover, berseem clover, and very occasionally white clover. These specific isoflavones are called formononetin, genistein, and biochanin A. As the Australians observed, subterranean clover can be the biggest culprit, with isoflavone levels that sometimes can reach nearly 5% of its dry weight. However — and this is a big however — isoflavone level is heavily influenced by genetics. Some varieties of subclover (and red clover, etc.) produce very high levels of isoflavones — like the classic Australian subclover varieties of Yarloop, Dinninup, and Tallarook — and some varieties produce very low levels. For example, we grow lots of subclover here in western Oregon, but our local varieties — Mt. Barker and Nangeela — are low in phytoestrogens, so we don't worry about grazing them with our sheep. And in Australia, as less and less acreage is being planted to high-isoflavone varieties, their incidence of clover disease is going down.

The other family of legume phytoestrogens — the coumestans — can occasionally occur in alfalfa (Medicago sativa) and the annual medics (other Medicago species). But I need to emphasize the word occasionally, because unlike isoflavone levels, which are highly correlated to genetics, coumestans seem to be a plant's response to leaf disease. These coumestans (specific compounds called coumestrol, sativol, and others) only appear in these plants after their leaves suffer serious stress, like fungus infections or attacks by aphids. Otherwise, these forages don't contain enough phytoestrogens to cause problems in livestock. This helps explain the sporadic reports with alfalfa — because the problem only occurs sporadically.

Phytoestrogens, particularly the isoflavones, create their reproductive havoc by affecting the cervix. In sheep after mating, sperm cells usually remain in the cervical area for a period prior to moving into the uterus. Normal cervical cells produce a mucous that helps store and support these sperm. Phytoestrogens alter this cervical mucous so that by 24 hours more than 95% of the sperm is no longer viable. Phytoestrogens also modify cervical cell growth, so that under a microscope the cervical tissue begins to look like a more like a uterus than a cervix. Cattle are also affected by phytoestrogens, but not quite in the same way. Fertility indeed declines, but cows show different symptoms such as cystic ovaries, irregular estrous cycles, etc.

But half the population can breathe a sigh of relief because these phytoestrogens have no effect on male reproduction. Rams and bulls remain fertile and virile, although an owner may become a bit unnerved to see udder development in a wether.

A point of interest, however, about dosage: molecule for molecule, legume phytoestrogens are actually far less potent than true mammalian hormones like estrogen. For example, estrogen is nearly 200x more potent then coumestrol, 6,900x more potent than genistein, and 26,500x more potent then formononetin. Livestock are affected by phytoestrogens because they can consume quite a lot of phytoestrogen molecules when they eat many pounds of forage.

But one nagging question comes to mind. Why do plants even make phytoestrogens? A clover plant certainly doesn't have a cervix, nor any need to regulate an estrous cycle. So why waste precious biochemical energy manufacturing useless compounds? But the concept of regulation may be a key to this riddle. Regulation means chemical communication — i.e. biological tissues communicating with each other. Cell-to-cell communication. When we look at the whole picture, a broader picture emerges: we see that the real role of estrogen is to communicate information (instructions) between different cells.

So which forage plants produce isoflavone phytoestrogens? Only legumes. Aha! Not grasses or trees or thistles. Legumes, of course, have that wonderful ability to create a symbiotic relationship with a specialized bacteria — Rhizobia species — that can extract ("fix") nitrogen from the air. These Rhizobia bacteria form nodules on the roots of legumes, where they create bacterial colonies and fix atmospheric nitrogen into bacterial protein. The legume plant not only gains nitrogen from this relationship; it also gains a great competitive advantage in the struggle for nutrients.

Each legume species needs its own unique Rhizobia species. So how, in that teeming, darkened world in the soil, can a plant get in touch with the appropriate bacteria? By communication. Possibly by using phytoestrogens. In fact, there are receptor proteins in Rhizobia bacteria that seem precisely designed for those phytoestrogens. This research is new, and it may not give an answer for all phytoestrogens, but scientists are beginning to speculate that when these bacterial proteins encounter the appropriate phytoestrogens, the resulting reaction tells the bacteria to initiate other biochemical processes which cause the ultimate formation of root nodules. The legume phytoestrogens, therefore, essentially act like signal lamps in a darkened room. "One if by land; two if by sea..."

The effects of phytoestrogens on livestock — clover disease, irregular estrous, lowered fertility — well, these problems may only be collateral damage from the main event, a chemical war between plants for nutrients and livelihood. Our sheep and cattle are just innocent bystanders.